title: Cisplatin-resistant neuroblastoma cells express enhanced levels of epidermal growth factor receptor (EGFR) and are sensitive to treatment with EGFR-specific toxins.
abstract: OBJECTIVE
  Neuroblastomas frequently show expression of the epidermal growth factor receptor (EGFR) and may therefore be susceptible to EGFR-targeted therapies. Here, EGFR expression and functionality was investigated in parental chemosensitive neuroblastoma cell lines (UKF-NB-3, IMR-32, NLF, SH-SY5Y) and their cisplatin-resistant sublines (UKF-NB-3(r)CDDP(1000), IMR-32(r)CDDP(1000), NLF(r)CDDP(1000), and SH-SY5Y(r)CDDP(500)). Moreover, the EGFR antibody cetuximab, the EGFR tyrosine kinase inhibitor Tyrphostin B46, and recombinant EGFR-targeted toxins were investigated for their influence on the viability and growth of neuroblastoma cells.
  
  
  METHODS
  EGFR expression and function was measured by flow cytometry or Western blot. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was examined by immunostaining for active caspase-3 or cleaved poly(ADP-ribose) polymerase. Cellular binding of FITC-labeled immunotoxins was studied by flow cytometry, and cellular uptake was studied by confocal laser scanning microscopy.
  
  
  RESULTS
  The EGFR-targeted antibody and growth factor toxins scFv(14E1)- Pseudomonas exotoxin A (ETA) and TGF-alpha-ETA exerted anti-cancer effects in neuroblastoma cell lines that were insensitive to cetuximab or EGFR tyrosine kinase inhibitors. Furthermore, adaptation of chemosensitive neuroblastoma cells to cisplatin increased EGFR expression and sensitivity to both recombinant toxins. Treatment of chemosensitive neuroblastoma cells with cisplatin reversibly increased EGFR expression, whereas cisplatin-resistant cells showed enhanced EGFR expression independent of the presence of cisplatin. Combination treatment with scFv(14E1)-ETA or TGF-alpha-ETA and cisplatin exerted significantly improved anticancer effects compared with either single treatment in parental neuroblastoma cells, cisplatin-resistant sublines, and primary cultures.
  
  
  CONCLUSIONS
  EGFR-targeted cytotoxic reagents such as scFv(14E1)-ETA and TGF-alpha-ETA represent promising candidates for further development as antineuroblastoma agents, especially in combination with cisplatin.
authors.affiliation: Institut für Medizinische Virologie, Klinikum der J.W. Goethe Universität, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Michaelis
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bliss
authors.forename: Sonja C
authors.initials: SC
authors.lastname: Arnold
authors.forename: Nora
authors.initials: N
authors.lastname: Hinsch
authors.forename: Florian
authors.initials: F
authors.lastname: Rothweiler
authors.forename: Hedwig E
authors.initials: HE
authors.lastname: Deubzer
authors.forename: Olaf
authors.initials: O
authors.lastname: Witt
authors.forename: Klaus
authors.initials: K
authors.lastname: Langer
authors.forename: Hans W
authors.initials: HW
authors.lastname: Doerr
authors.forename: Winfried S
authors.initials: WS
authors.lastname: Wels
authors.forename: Jindrich
authors.initials: J
authors.lastname: Cinatl
mesh_terms: D036002:ADP Ribose Transferases / Q000627:therapeutic use*; D000911:Antibodies, Monoclonal / Q000009:adverse effects; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001427:Bacterial Toxins / Q000627:therapeutic use*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D000068818:Cetuximab; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005098:Exotoxins / Q000627:therapeutic use*; D005434:Flow Cytometry; D006801:Humans; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000009:adverse effects; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use; D016211:Transforming Growth Factor alpha / Q000008:administration & dosage / Q000235:genetics; D014407:Tumor Cells, Cultured; D020032:Tyrphostins / Q000009:adverse effects; D037521:Virulence Factors / Q000627:therapeutic use*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D053148:Caspase 3; D000068818:Cetuximab; D002945:Cisplatin; D066246:ErbB Receptors; D005098:Exotoxins; D011065:Poly(ADP-ribose) Polymerases; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D020032:Tyrphostins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C095713:alpha-cyano-(3,4-dihydroxy)-N-(3-phenylpropyl)cinnamide